Eukaryotic gene expression is regulated by events at the levels of transcription, post-transcriptional processing, translation and stability of the mature mRNA. The removal of introns from the primary transcript is an essential step, and the use of alternative splicing patterns is one mechanism by which introns participate in the regulation of gene expression. The presence of some introns stimulate genes of mammals and their viruses. (C. J. Lai and G. Khoury (1979) Proc. Natl. Acad. Sci. 76:71-75; D. H. Hamer et al. (1979) Cell 18:1299-1302; P. Gruss and G. Khoury (1980) Nature 286:634-637; C. S. Gasser et al. (1982) Proc. Natl. Acad. Sci. 79:6522-6526; A. R. Buchman and P. Berg (1988) Mol. Cell. Biol. 8:4395-4405; and M. J. Evans and R. C. Scarpula (1989) Gene 84:135-142.) More recently, certain plant introns were shown to increase expression of homologous or heterologous genes in monocot and dicot species (J. Callis et al. (1987) Genes Develop. 1:1183-1200 and E. M. Silva et al. (1988) Genome 30(1):72). The identification and characterization of DNA elements that increase gene expression is essential to optimize expression of introduced genes in host cells, particularly transformed plant cells.
The shrunken-1 (Sh1) locus of maize encodes the major endosperm sucrose synthase, an enzyme important in the synthesis of starch. Loss of this enzymic activity results in inadequate starch levels and, in turn, in shrunken or collapsed kernels at maturity. Because of the gene's importance in starch metabolism, its very abundant transcript, its pivotal role in the study of transposable elements, and the extent of natural variation within the gene, the Sh1 locus has been cloned, its structure elucidated, and DNA sequence determined. (W. Werr et al. (1985) EMBO J. 4:1373-1380; H. Sheldon et al. (1983) Mol. Gen. Genet. 190:421-426; and C. D. Zack et al. (1986) Maydica. 31:5-16.)
The Sh1 locus is large and complex. The gene is composed of 16 exons and is 6 kbp in length. A relatively large intron of over 1000 base pairs separates the first and second exons. Although little is known about the function of such introns, it has been hypothesized that they may function to create new proteins by moving exons and/or changing rates of recombination within genes. (W. Gilbert (1978) Nature 271:501.)
Callis et al. (1987), supra, reported that introns from the maize genes Adh1 and bronze (Bz) increased gene expression in constructs similar to those used in the experiments reported here. Furthermore, Silva et al. (1988), supra, showed that the intron from a heat shock gene of maize increased expression of a reporter gene 3 to 6 fold when the 35S promoter was used to promote transcription.
While several maize introns apparently function to increase gene expression, the extent of activation is quite variable and is also dependent on the sequences being used to promote transcription. Previous studies have shown that the cauliflower mosaic virus 35S (CaMV 35S) promoter is 10-40 fold more effective than the nopaline synthase (NOS) promoter in driving the expression of foreign genes introduced into plant cells (M. Fromm et al. (1985) Proc. Natl Acad. Sci. USA 82:5824-5828; and R. M. Hauptmann et al. (1987) Plant Cell Rep. 6:265-270). These studies also demonstrated that the CaMV 35S promoter was 10-100 percent less effective in grass cells than in dicotyledonous species such as petunia and carrot. In our attempts to obtain efficient expression of introduced genes, we used promoters of the alcohol dehydrogenase-1 and -2 (Adh1, Adh2) and Sh1genes of maize (R. M. Hauptmann et al. (1988) Plant Physiol. 88:1063-1066). The Adh2 and Sh1 promoters gave no expression in grass protoplasts, while the levels of expression obtained with the Adh1 promoter were approximately 30% of the CaMV 35S promoter.
It has not been possible to predict with certainty which introns will increase expression and under what circumstances. In fact, some studies indicate that certain introns are not involved in gene expression. For example, a 1986 study found that the gene for the seed storage protein phaseolin was expressed at similar levels with or without its introns (P. P. Chee et al. (1986) Gene 41:47-57). Also, Callis et al. (1987), supra, showed that while the Adh1 first intron increased expression 16 to 112 fold when the Adh1 promoter was used, it increased expression only 5 to 22 fold in constructs containing the 35S promoter.
The present invention is based on a detailed analysis of the 5' untranslated region of the Sh1gene which has in part been described by V. Vasil et al. (1989) Plant Physiol. 91:1575-1579, incorporated by reference in its entirety herein.